1. Field of the Invention
The present invention relates generally to the field of immunology. More specifically, the present invention relates to chimeric antigen-enterotoxin mucosal immunogens that differentially enhance immune responses.
2. Description of the Related Art
Cholera toxin (CT) produced by Vibrio cholerae and the labile toxins (LT) from Escherichia coli are structurally related heat-labile enterotoxins (HLE) that have been employed as mucosal adjuvants to augment mucosal immune responses to co-administered antigens (Ag) (1, 2). These enterotoxins consist of an ADP-ribosylating A1 subunit non-covalently coupled with a pentameric ring of five identical B subunits through the A2 subunit, which is the C-terminal end of the A polypeptide (3). Initial studies using heat-labile enterotoxins as adjuvants in animal models led to the conclusion that their adjuvanticity was due to their toxic enzyme activity (4). ADP-ribosylation of the Gsα subunit of adenylate cyclase results in abnormally high levels of intracellular cAMP (3, 5), and subsequent chloride ion efflux into the lumen of the gut that is ultimately responsible for the characteristic watery diarrhea.
Due to the toxic nature of the holotoxins, many investigators have tried to dissociate the toxicity associated with the A1-subunit from the adjuvanticity of the AB5 complex, and have attempted to address the immunostimulatory effects of B5 subunit receptor-mediated interactions. Earlier studies using commercial CTB preparations contaminated with intact CT made it impossible to distinguish between the adjuvanticity associated with ADP-ribosyltransferase activity and the binding properties of the AB5 complex. This issue was further complicated by the synergistic effect of holotoxin on the adjuvanticity of the B subunit (6, 7). Experiments using a type I LT B subunit (LT-I B) mutant that lacks GM1-binding demonstrated that both immunogenicity and adjuvanticity were dependent upon receptor binding (8). Moreover, it has been demonstrated that upregulation of the co-stimulatory molecules, B7-1 and B7-2, on antigen-presenting cells (APC) by LT-I B or non-toxic derivatives of CT was abrogated when GM1 binding was blocked (9, 10). These studies demonstrate that GM1-B5 subunit interactions are necessary for the adjuvanticity associated with LT-I B or CT. However, LT-I and the type II HLE bind a broader range of gangliosides than CT, and it is not known whether these add to or substitute for GM1 binding in the immunostimulatory properties displayed by these molecules.
Two types of heat-labile enterotoxin have been distinguished on the basis of distinct immunoreactivity (11, 12): type I heat-labile enterotoxins are represented by CT and LT-I (12, 13); type II heat-labile enterotoxins include E. coli LT-IIa and LT-IIb (14-17). Comparison of the predicted amino acid sequences reveals considerable variability between type I and type II enterotoxins (11, 12, 18, 19). This extensive diversity imparts different ganglioside-binding properties to the respective B subunits. The cellular receptor for CT has been shown to be the monosialoganglioside GM1 (20). The B subunit of LT-IIa binds with high affinity to GD1b and less strongly to GM1, GT1b, GD1b, GD2, GD1a and GM2. LT-IIb binds with high affinity to GD1a (20).
It has recently been shown that the type II heat-labile enterotoxins can serve as mucosal adjuvants that enhance Ab responses to a co-administered protein antigen (Ag) given by the intranasal (i.n.) route (21). However, it is not clear if the different response patterns observed were related to the more promiscuous binding activities of the B subunits of the LT-II toxins, compared to CT. In addition to an adjuvant effect for co-administered Ag, heat-labile enterotoxins are themselves excellent mucosal immunogens and elicit strong secretory IgA and circulating IgG Ab responses. This property has been exploited in the construction of potent mucosal immunogens by coupling protein Ags to the non-toxic B subunit of CT (22-24). Initially, proteins were coupled to CTB by chemical conjugation, but later a genetic strategy for fusing an antigenic polypeptide to the A2 subunit of CT and co-expressing this with CTB was devised, yielding a chimeric immunogen in the form of Ag-CTA2/B (24, 25). Given that LT-II and CT have distinct ganglioside-binding specificity and somewhat different adjuvant activity, it is postulated that a chimeric protein consisting of Ag-LT-IIA2/B would have advantageous immunogenic properties compared to those of a chimeric protein consisting of Ag-CTA2/B. Therefore, the purpose of the present study was to compare the mucosal immunogenicity of chimeric proteins composed of the same protein Ag genetically coupled in an identical manner to the A2/B subunits of CT or LT-II. Possible mechanisms that might account for differences in their immunogenic properties were also explored.
The prior art is deficient in an effective means of enhancing immune responses by chimeric antigen-enterotoxin mucosal immunogens. The present invention fulfills this long-standing need and desire in the art.